Molecular insights into Spindlin1-HBx interplay and its impact on HBV transcription from cccDNA minichromosome

Molecular interplay between host epigenetic factors and viral proteins constitutes an intriguing mechanism for sustaining hepatitis B virus (HBV) life cycle and its chronic infection. HBV encodes a regulatory protein, HBx, which activates transcription and replication of HBV genome organized as covalently closed circular (ccc) DNA minichromosome. Here we illustrate how HBx accomplishes its task by hijacking Spindlin1, an epigenetic reader comprising three consecutive Tudor domains. Our biochemical and structural studies have revealed that the highly conserved N-terminal 2–21 segment of HBx (HBx2–21) associates intimately with Tudor 3 of Spindlin1, enhancing histone H3 “K4me3-K9me3” readout by Tudors 2 and 1. Functionally, Spindlin1-HBx engagement promotes gene expression from the chromatinized cccDNA, accompanied by an epigenetic switch from an H3K9me3-enriched repressive state to an H3K4me3-marked active state, as well as a conformational switch of HBx that may occur in coordination with other HBx-binding factors, such as DDB1. Despite a proposed transrepression activity of HBx2-21, our study reveals a key role of Spindlin1 in derepressing this conserved motif, thereby promoting HBV transcription from its chromatinized genome.

minichromosome. They show that HBx accomplishes its task by hijacking the epigenetic reader Spindlin1. Through biochemical and structural studies, they show that the N-terminal 2-21 segment of HBx (HBx 2-21) binds Tudor 3 domain of Spindlin1 that this interaction is accompanied by the switch from the repressive H3K9me3 mark to the H3K4me3 mark of active chromatin and promotes transcription from the cccDNA. The work is well conceived, and the AA present a set of high-quality results. However, the AA fail to clarify several important points: i) how the interaction between HBx and Spindlin-1 relates with the well-established activity of HBx (binding to DDB1 to redirect the DDB1 contaning E3 ubiquitin ligase complex to degrade Smc 5/6). Is HBx-Spindlin1 interaction down stream or upstream HBx activity on Smc 5/6? or, in other words, is HBx-Spindlin1 interaction required for HBx binding/activity on DDB1, or vice versa? ii) these considerations lead to another important question. From Figure 5, it appears that the effect on HBV transcription/replication of abrogating Spindlin1 activity is, to say the least, partial. In Figure 5G, the AA show that the Spindlin1 binding to the HBV cccDNA is well preserved in cells infected by the HBV HBx-mutant. Thus, the contribution of Spindlin1 to HBV transcription is there but modest and HBx is not required for Spindlin1 binding to the cccDNA. These considerations do not question the main message of the paper that HBx interaction with Spindlin1 has some effect on HBV cccDNA transcription. However, the AA must put their observations in the context of all other mechanisms that have been reported, repeatedly, to affect the epigenetic status of the HBV minichromosome and cccDNA transcription and to do so by generating novel experimental data. iii) the AA do not provide any direct mechanistic insight on how the interaction between HBx and Spindlin1 promotes the switch from the repressive H3K9me3 mark to the H3K4me3 mark of active chromatin on the cccDNA-bound histones. Finally, all functional data are obtained in HBV-infected HepG2-NTCP cells. The AA must confirm the observations (and their robustness) in HBV-infected primary human hepatocytes. As a minor point, the AA state "HBx interacts with Bcl-xL through the BH3-like domain to promote HBV production". It is unclear how this interaction relates to HBx activity on cccDNA transcription.
Reviewer #3 (Remarks to the Author): Spindlin1, an epigenetic reader and transcriptional coactivator, has previously be shown to interact with the HBV X protein (HBx), thereby modulating HBV gene transcription. However, the underlying mechanism was not clearly defined. In this manuscript, Liu et al. provide evidence that the host protein Spindlin1, can directly interact with an N-terminal motif within HBx. This interaction appears to facilitate binding to HBV cccDNA to promote HBV transcription in an H3K4me3K9me3-dependent manner. Using biochemical assays the authors explored systematically the interplay between Spindlin1 and HBx and confirmed their findings using x-ray crystallography. Overall, this is a well-designed study which provides new insights into how HBV hijack host factors, here spindling-1 to sustain establish and maintain persistence through epigenetic regulation. The data are clearly presented and largely support the authors' conclusions. However, there are a few concerns and open questions regarding the interplay between Spindlin1-HBx and how this complex interacts with cccDNA that need be further addressed.
Major comments • The authors have mapped amino acids that appear to be important for Spindlin1 and HBx binding (Figures 2&3) in vitro. It will be helpful to test the binding affinity between Spindlin1V232R, Spindlin1I245R, SpindlinK216S/V218R and HBx inside of the cells, particularly in the hepatic cells (for instance HepG2 or Huh7). • For the Spindlin1-HBx interaction with cccDNA part: The authors need to validate that the HBV DNA enriched with an anti-Spindlin1 is primarily cccDNA, and not HBV rcDNA, dslDNA or even integrated HBV DNA? Did the authors perform a T5 digestion step after CHIP and before qPCR? • Which region of the HBV genome does the Spindlin1-HBx complex bind to? Is it BCP region or other HBV transcription regulation regions like En 1/2? • How conserved the region within HBx (2-21) which appears to facilitate binding to Spindlin1 across different HBV genotypes? • in figure 1f, the bands for HBX2-21M are larger than that for WT. Did the authors compare the molecular weight of these two proteins to confirm this? • For the ChIP experiments in figure 4e, it would be important to also show the DNA gel and Sanger sequencing data to confirm the protein-DNA binding. • Line 144, style avoid using "interestingly", rather use notably or similar • line 159, "hepatocytes" rather than "hepatocyte" • Spindlin1 is thought to be a proto-oncogene which can promote oncogenic transcription and cell proliferation. Have the authors tested Spindlin1 knockdown has any effects on the cell cycle? Would the authors expect any effects on non-dividing (primary) hepatocytes

Reviewer #1
Remarks to the Author: Liu and colleagues report the identification of a novel HBV effector protein interaction motif that binds to the transcription regulator spindling 1 and modulates HBV transcription. In a series of elegant experiments the authors first map the interaction site on HBx with spindlin 1, then determine the crystal structure of the complex, and dissect the structural basis of the interaction. Then they show the functional conseqences of this interaction, and mechanism of action. Overall I very much enjoyed the manuscript, and the line of enquiry pursued by the time. I only have a few minor comments to make: Authors' response: We thank this reviewer for his/her positive comments by stating that "Overall I very much enjoyed the manuscript, and the line of enquiry pursued by the time." The different binding modes between spindoc and HBx are intriguing, and should be discussed in a little more detail. Are there significant differences in the precise amino acids that contact the spindling binding groove between the two interacting peptides? Could the authors speculate as to why the modes are so different?
Authors' response: Thanks for the comments. Structural alignment has revealed that HBx2-21 interacts with Tudor 3 of Spindlin1 in an opposite N to C orientation compared to that of SPINDOC256-281 (Fig. 2e). Despite this, both HBx2-21 and SPINDOC256-281 complete the β-barrel fold of Tudor 3 with similar hydrophobic core formation and β-sheet formation ( Figure R1a). This highlights both the conservation and diversity of Spindlin1's Tudor 3-mediated partner engagement.
Upon complex formation, HBx2-21 triggered minimal conformational change of Tudor 3, while SPINDOC256-281 induced more pronounced structural rearrangement around β strands 1 and 2 of Tudor 3 ( Figure R1b, c). One reason for the observed difference is the unique extended N-terminal motif of SPINDOC256-281, starting from F256, which contributes to binding ( Figure  R1c). We have included this detail discussions in our revised manuscript as new Supplementary  Fig. 2.  or PDB ID 5B1Z).

Figure R1. Structural comparison of Spindlin1-HBx2-21 complex with Spindlin1-SPINDOC256-281 complex. (a) Both HBx2-21 (light magenta) and SPINDOC256-281 (cyan) complete the β-barrel fold of Tudor 3 with similar hydrophobic core formation and β-sheet formation. (b) Structural alignment of free Spindlin1 Tudor 3 (green) (PDB ID 4MZF) and
For all SPR and ITC interactions shown, please state in the appropriate figure legend how often each measurement was performed for error quantification.
Authors' response: Thanks for the comments. An ITC curve typically involves more than 10 successive titrations (17 in this paper). The binding Kd and other thermodynamic parameters are calculated by fitting the titration curve using nonlinear regression. In this process, each data point represents an independent measurement of protein/ligand concentrations and a set of thermodynamic parameters, and the standard error of each parameter (e.g. Kd) will be estimated based on curve fitting over all data points (n=17 here) at 95% confidence. Similarly, for SPR analysis, signals were collected at one point/second (average of 8 images), and a total of 1,000 points were collected. The standard errors of binding parameters are calculated by fitting all data points as stated in the method section.

Minor issues:
Line 85: "a titling array of 20-mer" -overlapping array? Line 98: comparison Line 103: N-terminus of HBx Line 107: determine the molecular basis or gain molecular insight into Authors' response: Corrected. Thanks a lot! Line 112: The authors state that "HBx2-21 is intimately associated with a hydrophobic groove of Spindlin1 with high degree of shape complementarity". Please report a value for shape complementarity to support this statement, or clarify how shape complementarity was measured.
Authors' response: Thanks for the suggestion. The revised manuscript has included Sc (surface complementarity) score to quantitively describe the shape complementarity between HBx and Spindlin1. We calculated a Sc score of 0.7, which is even higher than that of antibody-antigen interfaces (where Sc ranges from 0.64 to 0.68) (Lawrence and Colman, 1993)"   Line 122: is also warranted -is supported?

Line 313: on a large scale -?
Authors' response: Thanks, we have rephrased this sentence to "Peptide concentrations were determined by weighing in large quantities".
Line 574: Data represent the mean±SD -how many measurements were performed (n=?) Authors' response: Thanks for the comment. The data was presented as mean±SE (standard error), which is estimated by nonlinear regression of the titration curve at 95% confidence. The data points used for fitting is 16 (n=16).

Reviewer #2
Remarks to the Author: Wei Liu and colleagues investigate the mechanism by which the HBV regulatory protein HBx activates transcription and replication of the viral nuclear covalently closed circular (ccc) DNA minichromosome. They show that HBx accomplishes its task by hijacking the epigenetic reader Spindlin1. Through biochemical and structural studies, they show that the N-terminal 2-21 segment of HBx (HBx 2-21) binds Tudor 3 domain of Spindlin1 that this interaction is accompanied by the switch from the repressive H3K9me3 mark to the H3K4me3 mark of active chromatin and promotes transcription from the cccDNA. The work is well conceived, and the AA present a set of high-quality results.
Authors' response: We thank this reviewer for his/her positive comments by stating that "The work is well conceived, and the AA present a set of high-quality results."

However, the AA fail to clarify several important points: i) how the interaction between HBx and Spindlin-1 relates with the well-established activity of HBx (binding to DDB1 to redirect the DDB1 contaning E3 ubiquitin ligase complex to degrade Smc 5/6). Is HBx-Spindlin1 interaction down stream or upstream HBx activity on Smc 5/6? or, in other words, is HBx-Spindlin1 interaction required for HBx binding/activity on DDB1, or vice versa?
Authors' response: Thanks for raising this important question. HBx binds to DDB1 through an H-box motif, HBx88-100, which is distinct from HBx2-21. Therefore, Spindlin1 and DDB1 do not directly compete for HBx binding. However, the predicted structure of HBx by AlphaFold (Jumper et al., 2021) shows that elements HBx2-21, HBx88-100 and HBx104-135 cluster to form a hydrophobic core ( Figure R3a). Interestingly, in the folded HBx structure, key residues involved in Spindlin1, DDB1 and Bcl-2 binding, such as M5, L16, L18 of HBx2-21, L89, L93, R96 of HBx88-100 and V116, W120, L123, I127, R128 of HBx110-135, are buried upon hydrophobic core formation ( Figure R3a). This suggests that effective engagement of HBx with DDB1 and Spindlin1 requires unfolding of HBx to expose key binding motifs. In this case, we speculate that HBx may exist in two conformational states: a folded inactive state and an extended active state ( Figure R3b). Conceivably, Spindlin1, DDB1 and Bcl-2 may cooperate with each other to interact with HBx by jointly overcoming the folding energy barrier, thus enabling a functional switch of HBx from an inactive state to an active one.
To experimentally investigate the functional correlation between Spindlin1 and DDB1, we conducted HBx pulldown and SMC6 degradation assays in HepG2-NTCP cells. We showed that siRNA knockdown of Spindlin1 resulted in slightly decreased level of DDB1 that binds to HBx ( Figure R3c). In SMC6 degradation assays, our western blot analysis revealed a significant decrease in the SMC6 level in cells expressing HBx. Surprisingly, such a decrease was partly restored in Spindlin1 knockdown cells ( Figure R3d). These observations are consistent with our structural analysis above, and support a positive crosstalk between Spindlin1-HBx and DDB1-HBx engagements as well as DDB1-HBx-mediated SMC5/6 degradation. We have included these new results in the revised manuscript as new Fig.7. ii) these considerations lead to another important question. From Figure 5, it appears that the effect on HBV transcription/replication of abrogating Spindlin1 activity is, to say the least, partial. In Figure 5G, the AA show that the Spindlin1 binding to the HBV cccDNA is well preserved in cells infected by the HBV HBx-mutant. Thus, the contribution of Spindlin1 to HBV transcription is there but modest and HBx is not required for Spindlin1 binding to the cccDNA. These considerations do not question the main message of the paper that HBx interaction with Spindlin1 has some effect on HBV cccDNA transcription. However, the AA must put their observations in the context of all other mechanisms that have been reported, repeatedly, to affect the epigenetic status of the HBV minichromosome and cccDNA transcription and to do so by generating novel experimental data.

Figure R3. Crosstalk between Spindlin1-HBx and DDB1-HBx engagements. (a) AlphaFold model of free HBx (left) and structure model of HBx binding to Spindlin1 (light magenta-green), DDB1 (cyan-grey, PDB ID 3I7H) and Bcl-2 (slate-white, PDB ID 5FCG). (b) Schematic model of HBx transitioning from an inactive state to an active one by binding to
Authors' response: Thanks for the valuable suggestion. Our study on HBV infected HepG2-NTCP and primary human hepatocytes demonstrated that inhibiting Spindlin1 activity resulted in a significant reduction in HBV transcription and HBeAg levels ( Fig.5b-e, Supplementary  Fig.4a-f). Rescue experiments also revealed that the ectopic expression of wild type Spindlin1 can restore HBV transcription and HBeAg levels in Spindlin1 knockdown cells (Fig. 5f, g), indicating that Spindlin1 plays a crucial role in promoting HBV transcription. While our findings are reliable and reproducible, we acknowledge that the role of Spindlin1-HBx engagement on HBV transcription/replication represents only one of the many mechanisms involved in HBV regulation. We are pleased that our work has identified a previously uncharacterized mechanism, highlighting the regulatory complexity of HBV life cycle from the perspective of epigenetics and cccDNA minichromosome.
In Fig. 5g, our ChIP analysis revealed a clear signal of Spindlin1 on cccDNA in HepG2-NTCP cells infected with wild type HBV. In contrast, the enrichment of Spindlin1 was reduced when HBx-deficient HBV was used for infection, indicating that HBx plays a role in stabilizing Spindlin1 at the HBV minichromosome. However, the enrichment of Spindlin1 on cccDNA still exists compared to the anti-IgG group (~0.5% of input), suggesting that Spindlin1 could target the cccDNA minichromosome via an HBx-independent mechanism. In fact, this is consistent with our observation that Spindlin1 is a potent reader of the histone H3 "K4me3-K9me3" methylation pattern (Kd = 13.3 nM) that marks poised heterochromatic regions (Du et al., 2021;Zhao et al., 2020a). Our binding studies revealed that when Spindlin1 forms a complex with HBx, its binding to H3 "K4me3-K9me3" could be further enhanced from 13.3 nM to 3.8 nM (Fig. 4b). Importantly, as discussed early, in addition to recruitment, Spindlin1-HBx engagement also plays a critical role in switching the conformational state of HBx, thereby unlocking its function. In the revised manuscript, we have included a new Fig. 7 to explain these findings and provide more discussion to contextualize our new observations with other reported mechanisms. iii) the AA do not provide any direct mechanistic insight on how the interaction between HBx and Spindlin1 promotes the switch from the repressive H3K9me3 mark to the H3K4me3 mark of active chromatin on the cccDNA-bound histones.
Authors' response: Thank you for the suggestion. As previously discussed, we have proposed in the revised manuscript a mechanism for the conformational switch of HBx triggered by Spindlin1 and DDB1. This explains how Spindlin1-HBx interaction initiates a switch of the cccDNA chromatin state from a repressive to an active one.
In mammalian cells, the SET1/MLL complexes are the primary writers of the H3K4me3 mark. It has been reported that they can methylate H3K4 in the presence of H3K9me3 (Patel et al., 2014). Therefore, H3K4me3 can be enzymatically created in H3K9me3-rich regions to establish a bivalent H3 "K4me3-K9me3" methylation pattern for robust Spindlin1 recruitment.
In this process, engagement between Spindlin1 and HBx not only promotes Spindlin1 recruitment but also triggers a functional switch of HBx from organizing a repressive chromatin state to an open one by cooperatively recruiting activation-related factors such as DDB1, CBP/p300, or other HBV-specific transcription factors (TFs). Intriguingly, it has been well established that erasers of H3K9me2/3, such as KDM4 and KDM7 family members, can recognize H3K4me3 to remove H3K9me2/3 marks. This "read-erase" mechanism may function to resolve the H3 "K4me3-K9me3" bivalent state and establish a fully active chromatin state marked by H3K4me3 and hyperacetylation. We have added more discussions and an updated working model (new Fig. 7e) to illustrate these points in the revised manuscript. Further research will be conducted to experimentally investigate whether and how the above-mentioned factors, such as SET1/MLL, KDM4/7, CBP/p300, participate in the epigenetic reprogramming of cccDNA minichromosome initiated by Spindlin1-HBx engagement.

Finally, all functional data are obtained in HBV-infected HepG2-NTCP cells. The AA must confirm the observations (and their robustness) in HBV-infected primary human hepatocytes.
Authors' response: Thanks for the suggestion. While HepG2-NTCP cells are an excellent model for studying HBV infection, we acknowledge the importance of confirming the observations and their robustness in HBV-infected primary human hepatocytes. Therefore, we conducted Spindlin1 knockdown studies in HBV-infected primary human hepatocytes. As anticipated, knockdown of Spindlin1 ( Figure R4a) led to a significant reduction in HBV RNA levels ( Figure R4b) and HBeAg levels in the medium supernatant ( Figure R4c). These results are consistent with those obtained in HBV-infected HepG2-NTCP cells, indicating the crucial role of Spindlin1 in stimulating HBV transcription. These new results have been incorporated in the revised Supplementary Fig. 4d-f. Figure R4. Spindlin1 is required for HBV transcription in HBV-infected primary human hepatocytes. (a) The efficiency of Spindlin1 knockdown in HBV infected cells by RT-qPCR analysis. (b-c) Primary human hepatocytes were transfected with Ctrl siRNA or Spindlin1 siRNAs for 16h and then infected with HBV. At 11dpi, cells and culture medium were harvest and HBV RNA levels and HBeAg levels were analyzed by RT-qPCR (b) and ELISA (c), respectively.
As a minor point, the AA state "HBx interacts with Bcl-xL through the BH3-like domain to promote HBV production". It is unclear how this interaction relates to HBx activity on cccDNA transcription.